The human mouth is colonized by a microbial community of enormous complexity that plays a key role in human health and disease. Increasingly, it is recognized that the spatial organization of microbial communities in unique micro-niches may have to be explored at fine resolution to provide the basis for elucidating the rules of community assembly and function and to understand their role in disease. Although next-generation DNA sequencing technology and metagenomics have revolutionized the analysis of microbial communities, a major gap in our understanding is the lack of spatial information at the micron level,--information necessary to better characterize the interrelationships between members of a microbial community (the microbial interactome) and with host tissue. We propose to fill this gap through a novel collaboration between the Borisy lab at the Marine Biological Laboratory and the Dewhirst lab at the Forsyth Institute and Harvard Medical School. The Borisy lab has recently developed a Combinatorial Labeling and Spectral Imaging (CLASI) strategy for the simultaneous identification of tens of microbial taxa in a single microscopic image. The Dewhirst lab has carried out ground-breaking metagenomic studies of the human oral microbiota. An initial collaboration between the two laboratories, supported by an ARRA Challenge Grant, applied the CLASI strategy to the analysis of human dental plaque, a microbial biofilm, and led to a proof-of-principle publication that characterized the proximity relationships of 15 oral taxa. Building on this foundation, our proposed research will visualize and define the micron-scale biogeography of the oral microbiome structure and interactome for 9 oral niches: subgingival plaque, supragingival plaque, keratinized gingivae, hard palate, tongue dorsum, buccal mucosa, palatine tonsils, throat, and saliva. Employing an automated tile-scanning capability, it will document the architecture and interactome for subgingival microbes in periodontal disease, demonstrating the utility of the approach for examining other oral diseases. It will produce a validated set of FISH probes for the oral research community. By revealing the precise, micron-scale structure of the oral microbiome, this research will address fundamental mechanisms and principles of community dynamics. It will provide an unprecedented systems-level understanding for how members of the human oral microbiota are organized and it will lay the foundation for future studies examining the role of the normal microbial flora in both healthy and diseased mouths. It will impact the broader oral and microbial research communities by developing methods, analysis tools and probes. This project should lead to the identification of key bacterial interactions that may serve as novel targets for therapeutic intervention.